Tuberculosis (TB) kills about 1.5 million people globally every year, making it the leading cause of death by a bacterial pathogen. The lack of an effective TB vaccine or the development of protective immunity after TB infection suggests antimicrobial drugs may be our best chance for controlling this disease. Current therapies are prolonged and toxic, and drug resistance is on the rise. Thus the TB field is eager to identify new drugs that are less toxic as well as more efficacious against drug-resistant strains. The Mycobacterium tuberculosis (Mtb) proteasome is essential for this bacterium to cause lethal infections in animals; thus, we want to understand how proteasome function is linked to Mtb physiology as well as target the proteasome and its co-factors for drug development. The proteasome is a multi-subunit, barrel shaped complex that degrades proteins. We found that enzymes required for protein degradation include PafA, which attaches prokaryotic ubiquitin-like protein (Pup) onto substrates targeted for destruction, and Mpa, which is an ATPase chaperone that recognizes pupylated proteins and unfolds them for delivery into the Mtb proteasome core. Pup can also be removed from substrates by a highly unique protease called Dop. More recently, we discovered a new co-factor called PafE, which forms dodecameric rings that open the proteasome to degrade proteins in an ATP-independent manner. Interestingly, PafE does not require Pup or any other post-translational modification to recognize proteins for degradation. This proposal outlines experiments that will determine how proteins are selected for Pup/Mpa-dependent and PafE-dependent proteasomal degradation, and determine the roles of PafE-dependent substrates on Mtb physiology and pathogenesis.